Project Summary The CDC lists MDR Neisseria gonorrhoeae (Ng) as one of the three most urgent antibiotic resistance threats in the United States. A Gram-negative fastidious organism, Ng causes gonorrhea, the second-most prevalent sexually transmitted bacterial infection (STI) with >800,000 estimated cases in the United States annually. Left untreated, gonorrhea can cause pelvic inflammatory disease in women, leading to fallopian tube scarring and infertility or may disseminate, causing joint and skin manifestations. Once easily treatable, Ng has evolved resistance to nearly every antibiotic used to treat it, leaving a combination of azithromycin (AZM) and ceftriaxone (CTX) as the only currently available treatment option. Importantly, a cluster of cases was recently reported in Hawaii that was resistant to both AZM and CTX, highlighting the critical need for new therapeutics targeting antibiotic-resistant Ng infections. Bacterial translation is plagued by transcription errors, mRNA damage, and translational frameshifts, which can result in loss of the stop codon and non-stop ribosome complex formation, preventing the release of protein products and inhibiting further translation. Recovery of non-stop ribosome complexes is an essential process in bacteria mediated by trans-translation machinery found in every bacterial genome sequenced to- date. Mutations in trans-translation components affect viability or virulence in a wide variety of bacteria including Ng. In preliminary studies, we demonstrated that tetrazole-based compounds inhibit trans-translation, acting as potent antimicrobials against a range of pathogens, including Ng, with a key analog inhibiting >95% of Ng strains tested at extremely low concentrations (MIC90 = 0.27 ?g/mL). Photoaffinity labeling and biochemical experiments suggest that inhibition of trans-translation is mediated through compound binding to a novel site of Elongation factor Tu (EF-Tu; an essential component of protein synthesis and trans-translation) acting selectively against trans-translation over translation. Preliminary evaluation of these compounds demonstrated limited cytotoxicity and a responsive SAR, enabling optimization of potency and resulting in the identification of MBX-3808, the lead compound in this proposal. The objective of this Phase I application is to demonstrate that these trans-translation inhibitors are effective in animal models of gonorrhea infection and build a dataset that will drive the transition from Hit-to-Lead to Lead Optimization. Phase II will focus on lead optimization to produce a preclinical candidate suitable for IND- enabling studies. We will achieve the Phase I objectives through four specific aims: In Aim 1 we will chemically optimize the trans-translation inhibitor series for potency, selectivity and in vitro ADME properties, targeting chemical synthesis of >200 analogs. In Aim 2, we will identify analogs with ADME-T and potency properties suitable for in vivo testing, analyzing potency and in vitro properties for all materials generated in Aim 1. In Aim 3, we will confirm and further explore the mechanism and specificity of tetrazole inhibitor of trans-translation in Ng. In Aim 4, we will evaluate lead analogs for bioavailability, acute toxicity and efficacy in murine models of Ng infection.